J Mol
Cell
Cardiol
Serum
Uichi
24,
579-584
Interleukh
Ikeda,
(1992)
6 Levels
Become II&wctia
Elevated
In Acute
Fuji0 Obkawa, Yoshitane Seine, Keiji Yamam Tadashi Kasaharat, Tadashi Kawai*, Kazuyuki
Departments of Cardiology,
Myocardial
oto, Yusuke Shimada
Hid&a*,
*Clinical Pathology, and IMedical Biology and Parasitology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan
(Received 28 May 1991, accepted in revisedfom? 30 December 1991) U. IKEDA, F. OHKAWA, Y. SEINO, K. YAMAMOTO, Y. HIDAKA, T. KASAHARA, T. KAWAI, AND K. SHIMADA. Serum Interleukin 6 Levels Become Elevated In Acute Myocardial Infarction. Journal of Molmhr and Celfular Cardiology (1992) 24, 579-584. We have examined serum interleukin 6 (IL-6) levels in 12 patients with acute myocardial infarction (AMI). IL-6 levels became elevated in all patients, following the rise of serum creatine kinase (CK) activity. Peak IL-6 levels showed a good correlation with peak serum C-reactive protein (CRP) levels, while there was no direct relationship between peak IL-6 levels and peak CK activity. IL-6 mRNA was not detected in unstimulated “quiescent” rat cardiocytes cultured in serum-free medium, but its expression was induced by exposure of the cells to serum or ionomycin. These results show that IL-6 is synthesized in the myocardium and serum IL-6 levels become elevated in AMI, suggesting that IL-6 could affect the progression and/or healing processes of AMI. KEY WORDS:
Interleukin
6; Creatine
kinase;
mRNA;
Myocardial
Introduction In acute myocardial infarction (AMI), there is an accumulation of lymphocytes, monocytes and neutrophils in the infarcted area. Interleukin 6 (IL-6) is produced mainly by lymphocytes, monocytes, as well as by endothelial and vascular smooth muscle cells [I, 21. IL-6 has a wide variety of biological functions [I], such as the induction of terminal maturation of activated B cells and the regulation of the major acute phase protein response, however, the effects of IL-6 on the cardiovascular system, particularly in AMI, are not well known. In the present study, we have examined serum IL-6 levels in patients with AMI. IL-6 levels became elevated in these patients, and could affect the progression and/or healing processes of AMI. Methods Patients Serum IL-6 levels were examined ients with AM1 who were admitted Please wachi-Machi,
address
0022-2828/92/060579
all Tochigi
correspondence 329-04, Japan. + 06 $03.00/O
in 12 patto our hos-
to: Uichi
Ikeda,
infarction;
CRP.
pital within 6 hours after the onset of chest pain. The diagnosis of AM1 was based on persistent chest pain, electrocardiographic changes (ST segment changes andfor Q waves), and a rise in serum creatine kinase (CK) activity. Of 12 patients, seven were men and five were women, with an average age of 64 years (range, 48 to 82 years). Venous blood samples were collected every 4 hours during the first few days, and at least once a day thereafter during the 7 days after admission. The serum samples were stored at - 80% until measurements could be taken.
IL-6
measuremmts
Serum IL-6 levels were measured with a solidphase sandwich-immunoassay system (Fuji Rebio, Jpn) using a monoclonal anti-humanIL-6 antibody with a sensitivity of 2pg/ml. Measurements were performed according to the manufacture’s protocol. 50 pl of the serum sample were used for the measurements.
Department
of Cardiology,
Jichi
Medical
@ 1992
School,
Academic
Minamika
Press
Limited
580
U. Ikeda Cell culture
Primary cardiocytes from 1 -day-old SpragueDawley rats were prepared using the method of Bloch et al. [3] with minor modifications. After 0.4% trypsin dissociation, cell suspensions were washed with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), plated on 100 mm culture dishes and incubated for 2 h at 37%. During this interval, mesenchymal cells and a few myocytes attached to the culture dishes, while most myocytes remained suspended in the medium. The medium containing myocytes was pooled and transferred to 60mm dishes at a density of 4 x lo6 cells/dish in 10% FBS containing DMEM supplemented with thymidine (0.6 mg/ml), penicillin (20 U/ml), streptomycin (20pg/ml), and gentamycin (20pglml). After 48h incubation, the medium was changed to serum-free DMEM supplemented with insulin (5pg/ml), transferrin (5pg/ml), and selenium (5 rig/ml). Cells were used for experiments after 24 h preincubation in serum-free DMEM.
et al. extracted from myocytes by the guanidinium isothiocynate/caesium chloride procedure [ 41. Total RNA, lOpg, was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes (Hybond N + , Amersham, England). Filters were hybridized with a 32P-labelled murine IL-6 cDNA probe (specific activity > 1 x 10acpmlpg DNA) at 60% for 24h. The IL-6 probe consisted of a 1.4 kb EcoR I restriction fragment [5, 61. At the end of hybridization, the filters were washed twice in 0.2 x SSC at 60% (1 x SSC contains 0.15~ NaCl, 0.015M sodium citrate, pH 7.0). Filters were exposed to Kodak XAR-5 film for 2-3 days at - 70% with one intensifying screen. Miscellaneous Statistical analysis was performed using the Student’s t-test. P values less than 0.05 were considered to indicate a statistically significant difference. The murine IL-6 cDNA probe was a kind gift of Dr T. Sudo (Biomaterial Institute, Japan). Chemicals were of the highest commercially available grade.
Northern blot anaEyris
Results
After 24 h preincubation in serum-free DMEM, myocytes were exposed to 10% FBS or 10 -6 M ionomycin for 2 h. Total RNA was
2000
Figure 1 shows the case of a 57-year-old man with AM1 who was admitted to our hospital 4 h after the onset of chest pain. At admission,
-
i 3 J u’
IOOO-
O-
1234567
Hospital
FIGURE our hospital ( o ; CRP)
1. Serum IL-6, CK and CRP levels in acute myocardial 4h after the onset of chest pain. Creatine kinase (0; CK), in the serum were examined during 7 days after admission.
days
infarction. interleukin
A 57-year-old 6 (0; IL-6),
man was admitted to and C-reactive protein
IL-6inAMl
581
100 0 5
y = 3.23x f -0.10,
+ 21.9 P>O.O5
x ‘f =I ;: 5
500 a .
w
v
:
0
0
0 I 1000
0
Mox
FIGURE infarction,
0
I 2000 CK
(M/I)
2. Correlation between max serum IL-6 and max serum three on (o) and nine not on (0) thrombolytic therapy
CK levels in 12 patients
with acute myocardial
100
t
y -2.87x I = 0.64,
+ 5.74 PCO.05
5o-*
0
I 5
I IO Mox
FIGURE infarction,
CRP
1 15
(mg/dl)
3. Correlation betweem max serum IL-6 and max serum CRP levels in 12 patients three on (0) and nine not on (0) thrombolytic therapy.
an electrocardiogram revealed ST segment elevation in I and aVL leads. Serum CK activity was 86 IU/l(normal~70IU/l). A coronary angiogram revealed a stenotic lesion in the left circumflex artery, so intracoronary thrombolytic therapy with urokinase was performed. Serum CK activity reached peak values at 9 h. The serum IL-6 level increased following the elevation of CK activities; the level was within normal limits ((2 pg/ml) at
with acute myocardial
admission, began to rise at 9 h, and peaked at 28 h after symptom recognition. C-reactive protein (CRP), which is synthesized in the liver by stimulation of IL-6, rose gradually following the IL-6 increase. In all of the other 11 AM1 patients, IL-6 levels also began to rise within several hours after the onset of chest pain and continued to increase with a same or delayed time course to the rise of CK activity. Figure 2 shows peak IL-6 levels and peak
582
U. Ikeda et al. -6
FIGURE 4. IL-6 gene expression in myocardial cells. Cultured neonatal rat myocytes pretreated for 24h in serumfree DMEM were exposed to 10% fetal bovine serum (FBS) or 10-6M ionomycin for 2 h. Total RNA was extracted from the cells by the GITC/CsCI procedure, size-fractionated by electrophoresis and transferred to nylon membranes as described in the text. Filters were directly visualized by UV transillumination (RNA) or hybridized with a 32P-labelled IL-6 cDNA probe (IL-6). Lane 1; control, lane 2; 2 h exposure to 10 7% FBS, lane 3; 2 h exposure to 10 - 6 M ionomycin. Data are representative of three separate experiments.
CK levels in 12 patients, three on and nine not on thrombolytic therapy. There was no significant correlation between peak IL-6 and peak CK levels. On the other hand, there was a significant positive correlation between peak IL-6 and peak CRP levels (,y= 2.87x+5.74, r=0.64, PcO.05) (Fig. 3). Northern blotting experiments did not
reveal IL-6 transcript in RNA isolated from unstimulated “quiescent” myocytes cultured in serum-free medium for 24 h (Fig. 4). However, IL-6 mRNA was detected after 2 h exposure of myocytes to 10 % FBS or 10 -6 M ionomycin, although the response was more prominent in the former than the latter.
IL-6inAlW Discussion In a myocardial infarcted area, there is an accumulation of ueutrophils, lymphocytes, and monocytes. These cells produce various kinds of active agents such as arachidonate lipoxygenase metabolites, proteolytic enzymes and oxygen free radicals under stimulated conditions [ 7, 81. IL-6 is one such agent, secreted mainly from lymphocytes, monocytes, as well as from endothelial and vascular smooth muscle cells [9]. In this study, we have shown that serum IL-6 levels become elevated in patients with AMI, and that myocardial cells can express IL-6 gene transcript in response to serum, or ionomycin which induces intracellular Ca2 + elevation as seen in ischaemic myocardium [ 101. Therefore, we speculate that IL-6 is secreted from lymphocytes, monocytes, vascular tissues and/or myocardial cells in the infarcted area and released into the systemic circulatory system. Our results show no direct relationship between peak IL-6 levels and peak values of serum CK activity. This can be explained by the fact that IL-6 levels are affected by various factors such as complications of infection and serum catecholamine levels [II]. On the other hand, we observed a positive correlation between peak IL-6 levels and peak CRP levels, compatible with the fact that CRP is produced in the liver mainly by stimulation of IL-6 [ 121. Recently, we have reported that IL-6 stimulates the growth of vascular smooth muscle cells in an autocrine manner 191, and Motro et al. have reported that IL-6 possesses angiogenic activity [ 131. However, the effects of IL-6 on the cardiovascular system, particularly in AMI, are not well known. Cytotoxic effects of IL-6 on certain cells such as myeloid leukaemic cell lines have been reported [12], but in our observation IL-6 showed no cyto-
583
toxic effects on cultured neonatal rat myocytes (data not shown). Involvements of interleukin 1 (IL-l) in AM1 have been suggested in several previous studies; (i) vadodilator effects of IL-1 on rat aortae or canine coronary arteries [I4, 151, and (ii) decreases in ischaemia/reperfusion injury in rat hearts by IL-1 pretreatment [16]. Considering that some biological functions of IL-1 are mediated by the production of IL-6 and these two interleukins have a broad spectrum of activities in common [I, 17j, it is reasonable to speculate that IL-6 also has effects on the cardiovascular system. Indeed, in our preliminary study, IL-6 inhibited the contraction of rat aortic ring induced by phenylephrine. Neutrophils apparently contribute to the injury of myocardial tissue after occlusion of the coronary artery, particularly under conditions where the ischaemic tissue is subsequently reperfused with normally oxygenated blood. Neutrophil attachment to vessel walls and migration into cardiac tissue begins shortly after the initiation of reperfusion, and recently Entman et al. [18] have reported that IL-6 promotes the adhesion of neutrophils to cardiac myocytes in vitro. In conclusion, serum IL-6 levels become elevated in AMI, suggesting that IL-6 could affect the progression and/or healing processes of AMI.
Acknowle&mcnts We thank E. Chiku and S. Tanaka for their technical assistance. This study was supported by the Kowa Life Science Foundation, the Takeda Medical Research Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders (Japan).
References 1 WONG, G. G., CLARK, S. C. Multiple actions of interleukin 6 within a cytokine network. Immunol Today 9, 137-139 (1988). 2 IKEDA, II., IKEDA, M., SEINO, Y., TAKAHASHI, M., KANO, S., SHIMADA, K. Interleukin 6 gene transcripts are expressed in atherosclerotic lesions of genetically hyperlipidemic rabbits. Atherosclerosis 92, 213-218 (1992). 3 BLOCH, K., SEIDMAN, J. G., NAF~ILAN, J. D., FALLON, J.T., SEIDMAN, C. E. Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways. Cell 47, 695-702 (1986). 4 CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., RUT~ER, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5298 (1979). 5 CHIU, C., MOULDS, C., COFFMAN, R. L., RENNICK, D., LEE, F. Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Nat1 Acad Sci USA 85, 7099-7103 (1988).
584
U. Ikcda
et al.
6 SNICK, V., CAYPHAS, J. S., SZIKORA, J,, RENAULD, J.. ROOST, E. V., BOON, T., SIMPSON, R. J. cDNA cloning of murine interleukin-HP1: homoloav with human interleukin 6. Eur I Immunol 18, 193-197 11988). 7 IKEDA, U., TOYO-OKA, T., HOSO;;;\, S. Stimulated synthesis of prostaglandin E, or leukotrien‘C, from myocardial cells is not a cause but a result of their injury under hypoxia. J Mol Cell Cardiol 19, 523-526 (1987). 8 EVERS, A. S., MURPHREE, S., SAFFITZ, J. E., JAKSCH~K, B.A., NEEDLEMAN, P. Effects of endogenously produced leukotrienes, thromboxane, and prostaglandins on coronary vascular resistance in rabbit myocardial infarction. J Clin Invest 75, 992-999 (1985). 9 IKEDA, U., IKEDA, M., OOHARA, T., OGUCHI, A., KAMITANI, T., TSURUYA, Y., KANO, S. Interleukin-6 stimulates the growth of vascular smooth muscle cells in a PDGF-dependent manner. Am J Physiol 280, H1713-H1717 (1991). 10 ALLEN, D. G., ORCHARD, C. H. Intracellular calcium concentration during hypoxia and metabolic inhibition in mammalian ventricular muscle. J Physiol (Lond) 339, 107-122 (1983). 11 GOOL, V., VUGT, H., HELLE, M., AARDEN, L. A. The relation among stress, adrenalin, interleukin 6 and acute phase proteins in the rat. Clin Immunol Immunopathol 57, 200-210 (1990). 12 HIRANO, T., AKIRA, S., TAGA, T., Biological and clinical aspects of interleukin 6. Immunol Today 11, 443-449 (1990). 13 MOTRO, B., ITIN, A., SACHS, L., KESHET, E. Patterns of interleukin 6 gene expression in viva suggests a role for this cytokine in angiogenesis. Proc Nat1 Acad Sci USA 87, 3092-3096 (1990). 14 BEASLEY, D., COHEN, R. A., LEVINSKY, N. G. Interleukin 1 inhibits contraction of vascular smooth muscle. J Clin Invest 88, 331-335 (1989). 15 KENNY, D., MCCARTHY-KENNY, G., PELC, L. R., CHEUNC, J. S., BROOKS, J. L., WARLTIER, D. C. Vasodilator actions of interleukin-1 in the canine coronary circulation. Basic Res Cardiol 82, 279-284 (1990). 16 BROWN, J. M., WHITE, C. W., TERADA, L. S., GROSSO,M. A., SHANLEY, P. F., MULVIN, D. W., BANERJEE, A., WHITMAN, G. R., HARKEN, A. H. Interleukin-1 pre-treatment decreases ischemia/reperfusion injury. Proc Nat1 Acad Sci USA 87, 5026-5030 (1990). 17 ONOZAKI, K., AKIYAMA, Y., OKANO, A., HIRANO, T., KISHIMOTO, T., HASHIMOTO, T., YOSHIZAWA, K., TANIYAMA, T., Synergistic regulatory effects of interleukin 6 and interleukin 1 on the growth and differentiation of human and mouse myeloid leukemic cell lines. Cancer Res 49, 3602-3607 (1989). 18 ENTMAN, M. L., YOUKER, K., SHAPPELL, S. B., SIEGEL, C., ROTHLEIN, R., DREYER, W. J. SCHMALSTIEG, F., SMITH, C. W. Neutrophil adherence to isolated adult canine myocytes: evidence for a CDlB-dependent mechanism. J Clin Invest 85, 1497-1506 (1990).
Cell
Cardiol
Serum
Uichi
24,
579-584
Interleukh
Ikeda,
(1992)
6 Levels
Become II&wctia
Elevated
In Acute
Fuji0 Obkawa, Yoshitane Seine, Keiji Yamam Tadashi Kasaharat, Tadashi Kawai*, Kazuyuki
Departments of Cardiology,
Myocardial
oto, Yusuke Shimada
Hid&a*,
*Clinical Pathology, and IMedical Biology and Parasitology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan
(Received 28 May 1991, accepted in revisedfom? 30 December 1991) U. IKEDA, F. OHKAWA, Y. SEINO, K. YAMAMOTO, Y. HIDAKA, T. KASAHARA, T. KAWAI, AND K. SHIMADA. Serum Interleukin 6 Levels Become Elevated In Acute Myocardial Infarction. Journal of Molmhr and Celfular Cardiology (1992) 24, 579-584. We have examined serum interleukin 6 (IL-6) levels in 12 patients with acute myocardial infarction (AMI). IL-6 levels became elevated in all patients, following the rise of serum creatine kinase (CK) activity. Peak IL-6 levels showed a good correlation with peak serum C-reactive protein (CRP) levels, while there was no direct relationship between peak IL-6 levels and peak CK activity. IL-6 mRNA was not detected in unstimulated “quiescent” rat cardiocytes cultured in serum-free medium, but its expression was induced by exposure of the cells to serum or ionomycin. These results show that IL-6 is synthesized in the myocardium and serum IL-6 levels become elevated in AMI, suggesting that IL-6 could affect the progression and/or healing processes of AMI. KEY WORDS:
Interleukin
6; Creatine
kinase;
mRNA;
Myocardial
Introduction In acute myocardial infarction (AMI), there is an accumulation of lymphocytes, monocytes and neutrophils in the infarcted area. Interleukin 6 (IL-6) is produced mainly by lymphocytes, monocytes, as well as by endothelial and vascular smooth muscle cells [I, 21. IL-6 has a wide variety of biological functions [I], such as the induction of terminal maturation of activated B cells and the regulation of the major acute phase protein response, however, the effects of IL-6 on the cardiovascular system, particularly in AMI, are not well known. In the present study, we have examined serum IL-6 levels in patients with AMI. IL-6 levels became elevated in these patients, and could affect the progression and/or healing processes of AMI. Methods Patients Serum IL-6 levels were examined ients with AM1 who were admitted Please wachi-Machi,
address
0022-2828/92/060579
all Tochigi
correspondence 329-04, Japan. + 06 $03.00/O
in 12 patto our hos-
to: Uichi
Ikeda,
infarction;
CRP.
pital within 6 hours after the onset of chest pain. The diagnosis of AM1 was based on persistent chest pain, electrocardiographic changes (ST segment changes andfor Q waves), and a rise in serum creatine kinase (CK) activity. Of 12 patients, seven were men and five were women, with an average age of 64 years (range, 48 to 82 years). Venous blood samples were collected every 4 hours during the first few days, and at least once a day thereafter during the 7 days after admission. The serum samples were stored at - 80% until measurements could be taken.
IL-6
measuremmts
Serum IL-6 levels were measured with a solidphase sandwich-immunoassay system (Fuji Rebio, Jpn) using a monoclonal anti-humanIL-6 antibody with a sensitivity of 2pg/ml. Measurements were performed according to the manufacture’s protocol. 50 pl of the serum sample were used for the measurements.
Department
of Cardiology,
Jichi
Medical
@ 1992
School,
Academic
Minamika
Press
Limited
580
U. Ikeda Cell culture
Primary cardiocytes from 1 -day-old SpragueDawley rats were prepared using the method of Bloch et al. [3] with minor modifications. After 0.4% trypsin dissociation, cell suspensions were washed with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), plated on 100 mm culture dishes and incubated for 2 h at 37%. During this interval, mesenchymal cells and a few myocytes attached to the culture dishes, while most myocytes remained suspended in the medium. The medium containing myocytes was pooled and transferred to 60mm dishes at a density of 4 x lo6 cells/dish in 10% FBS containing DMEM supplemented with thymidine (0.6 mg/ml), penicillin (20 U/ml), streptomycin (20pg/ml), and gentamycin (20pglml). After 48h incubation, the medium was changed to serum-free DMEM supplemented with insulin (5pg/ml), transferrin (5pg/ml), and selenium (5 rig/ml). Cells were used for experiments after 24 h preincubation in serum-free DMEM.
et al. extracted from myocytes by the guanidinium isothiocynate/caesium chloride procedure [ 41. Total RNA, lOpg, was size-fractionated by electrophoresis on 1.0% agarose gels, and transferred to nylon membranes (Hybond N + , Amersham, England). Filters were hybridized with a 32P-labelled murine IL-6 cDNA probe (specific activity > 1 x 10acpmlpg DNA) at 60% for 24h. The IL-6 probe consisted of a 1.4 kb EcoR I restriction fragment [5, 61. At the end of hybridization, the filters were washed twice in 0.2 x SSC at 60% (1 x SSC contains 0.15~ NaCl, 0.015M sodium citrate, pH 7.0). Filters were exposed to Kodak XAR-5 film for 2-3 days at - 70% with one intensifying screen. Miscellaneous Statistical analysis was performed using the Student’s t-test. P values less than 0.05 were considered to indicate a statistically significant difference. The murine IL-6 cDNA probe was a kind gift of Dr T. Sudo (Biomaterial Institute, Japan). Chemicals were of the highest commercially available grade.
Northern blot anaEyris
Results
After 24 h preincubation in serum-free DMEM, myocytes were exposed to 10% FBS or 10 -6 M ionomycin for 2 h. Total RNA was
2000
Figure 1 shows the case of a 57-year-old man with AM1 who was admitted to our hospital 4 h after the onset of chest pain. At admission,
-
i 3 J u’
IOOO-
O-
1234567
Hospital
FIGURE our hospital ( o ; CRP)
1. Serum IL-6, CK and CRP levels in acute myocardial 4h after the onset of chest pain. Creatine kinase (0; CK), in the serum were examined during 7 days after admission.
days
infarction. interleukin
A 57-year-old 6 (0; IL-6),
man was admitted to and C-reactive protein
IL-6inAMl
581
100 0 5
y = 3.23x f -0.10,
+ 21.9 P>O.O5
x ‘f =I ;: 5
500 a .
w
v
:
0
0
0 I 1000
0
Mox
FIGURE infarction,
0
I 2000 CK
(M/I)
2. Correlation between max serum IL-6 and max serum three on (o) and nine not on (0) thrombolytic therapy
CK levels in 12 patients
with acute myocardial
100
t
y -2.87x I = 0.64,
+ 5.74 PCO.05
5o-*
0
I 5
I IO Mox
FIGURE infarction,
CRP
1 15
(mg/dl)
3. Correlation betweem max serum IL-6 and max serum CRP levels in 12 patients three on (0) and nine not on (0) thrombolytic therapy.
an electrocardiogram revealed ST segment elevation in I and aVL leads. Serum CK activity was 86 IU/l(normal~70IU/l). A coronary angiogram revealed a stenotic lesion in the left circumflex artery, so intracoronary thrombolytic therapy with urokinase was performed. Serum CK activity reached peak values at 9 h. The serum IL-6 level increased following the elevation of CK activities; the level was within normal limits ((2 pg/ml) at
with acute myocardial
admission, began to rise at 9 h, and peaked at 28 h after symptom recognition. C-reactive protein (CRP), which is synthesized in the liver by stimulation of IL-6, rose gradually following the IL-6 increase. In all of the other 11 AM1 patients, IL-6 levels also began to rise within several hours after the onset of chest pain and continued to increase with a same or delayed time course to the rise of CK activity. Figure 2 shows peak IL-6 levels and peak
582
U. Ikeda et al. -6
FIGURE 4. IL-6 gene expression in myocardial cells. Cultured neonatal rat myocytes pretreated for 24h in serumfree DMEM were exposed to 10% fetal bovine serum (FBS) or 10-6M ionomycin for 2 h. Total RNA was extracted from the cells by the GITC/CsCI procedure, size-fractionated by electrophoresis and transferred to nylon membranes as described in the text. Filters were directly visualized by UV transillumination (RNA) or hybridized with a 32P-labelled IL-6 cDNA probe (IL-6). Lane 1; control, lane 2; 2 h exposure to 10 7% FBS, lane 3; 2 h exposure to 10 - 6 M ionomycin. Data are representative of three separate experiments.
CK levels in 12 patients, three on and nine not on thrombolytic therapy. There was no significant correlation between peak IL-6 and peak CK levels. On the other hand, there was a significant positive correlation between peak IL-6 and peak CRP levels (,y= 2.87x+5.74, r=0.64, PcO.05) (Fig. 3). Northern blotting experiments did not
reveal IL-6 transcript in RNA isolated from unstimulated “quiescent” myocytes cultured in serum-free medium for 24 h (Fig. 4). However, IL-6 mRNA was detected after 2 h exposure of myocytes to 10 % FBS or 10 -6 M ionomycin, although the response was more prominent in the former than the latter.
IL-6inAlW Discussion In a myocardial infarcted area, there is an accumulation of ueutrophils, lymphocytes, and monocytes. These cells produce various kinds of active agents such as arachidonate lipoxygenase metabolites, proteolytic enzymes and oxygen free radicals under stimulated conditions [ 7, 81. IL-6 is one such agent, secreted mainly from lymphocytes, monocytes, as well as from endothelial and vascular smooth muscle cells [9]. In this study, we have shown that serum IL-6 levels become elevated in patients with AMI, and that myocardial cells can express IL-6 gene transcript in response to serum, or ionomycin which induces intracellular Ca2 + elevation as seen in ischaemic myocardium [ 101. Therefore, we speculate that IL-6 is secreted from lymphocytes, monocytes, vascular tissues and/or myocardial cells in the infarcted area and released into the systemic circulatory system. Our results show no direct relationship between peak IL-6 levels and peak values of serum CK activity. This can be explained by the fact that IL-6 levels are affected by various factors such as complications of infection and serum catecholamine levels [II]. On the other hand, we observed a positive correlation between peak IL-6 levels and peak CRP levels, compatible with the fact that CRP is produced in the liver mainly by stimulation of IL-6 [ 121. Recently, we have reported that IL-6 stimulates the growth of vascular smooth muscle cells in an autocrine manner 191, and Motro et al. have reported that IL-6 possesses angiogenic activity [ 131. However, the effects of IL-6 on the cardiovascular system, particularly in AMI, are not well known. Cytotoxic effects of IL-6 on certain cells such as myeloid leukaemic cell lines have been reported [12], but in our observation IL-6 showed no cyto-
583
toxic effects on cultured neonatal rat myocytes (data not shown). Involvements of interleukin 1 (IL-l) in AM1 have been suggested in several previous studies; (i) vadodilator effects of IL-1 on rat aortae or canine coronary arteries [I4, 151, and (ii) decreases in ischaemia/reperfusion injury in rat hearts by IL-1 pretreatment [16]. Considering that some biological functions of IL-1 are mediated by the production of IL-6 and these two interleukins have a broad spectrum of activities in common [I, 17j, it is reasonable to speculate that IL-6 also has effects on the cardiovascular system. Indeed, in our preliminary study, IL-6 inhibited the contraction of rat aortic ring induced by phenylephrine. Neutrophils apparently contribute to the injury of myocardial tissue after occlusion of the coronary artery, particularly under conditions where the ischaemic tissue is subsequently reperfused with normally oxygenated blood. Neutrophil attachment to vessel walls and migration into cardiac tissue begins shortly after the initiation of reperfusion, and recently Entman et al. [18] have reported that IL-6 promotes the adhesion of neutrophils to cardiac myocytes in vitro. In conclusion, serum IL-6 levels become elevated in AMI, suggesting that IL-6 could affect the progression and/or healing processes of AMI.
Acknowle&mcnts We thank E. Chiku and S. Tanaka for their technical assistance. This study was supported by the Kowa Life Science Foundation, the Takeda Medical Research Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders (Japan).
References 1 WONG, G. G., CLARK, S. C. Multiple actions of interleukin 6 within a cytokine network. Immunol Today 9, 137-139 (1988). 2 IKEDA, II., IKEDA, M., SEINO, Y., TAKAHASHI, M., KANO, S., SHIMADA, K. Interleukin 6 gene transcripts are expressed in atherosclerotic lesions of genetically hyperlipidemic rabbits. Atherosclerosis 92, 213-218 (1992). 3 BLOCH, K., SEIDMAN, J. G., NAF~ILAN, J. D., FALLON, J.T., SEIDMAN, C. E. Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways. Cell 47, 695-702 (1986). 4 CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., RUT~ER, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5298 (1979). 5 CHIU, C., MOULDS, C., COFFMAN, R. L., RENNICK, D., LEE, F. Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Nat1 Acad Sci USA 85, 7099-7103 (1988).
584
U. Ikcda
et al.
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